We have undertaken the following projects: Anthrax studies Aims: 1) To determine whether or not the spin-trapping agent, DMPO, can alter the course of anthrax lethal toxin intoxication. 2) To probe the differential protein expression in macrophages as a result of lethal toxin challenge. Results: WE found that an impurity in DMPO was the active agent in prevention of LT intoxication. We have not been able to identify this compound. 2)The macrophage exposure study has been completed and the protein extracts are in the process of characterization using MSe based label-free identification and quantitation. Mass Spectrometry in Neurodegenerative Diseases. Aim: To characterize the O-GlcNAc modification in the cytoplasmic APP fragment using immuno-analytical techniques and mass spectrometry. Following the identification of O-glycosylation sites in recombinant APP695, we decided to characterize the O-glycosylation of APP isolated from the brain of CRND8 mice and their wild type littermates. CRND8 mice express an aggressive phenotype of AD, as a result of simultaneous Swedish (K670N/M671L) and Indiana (V717F) mutations in the APP gene. to investigate whether the C-terminal APP fragment (649-695) with cellular localization in the full-length APP695, represents an in vitro substrate for human O-GlcNAc transferase (OGT). The first part of this aim is to identify O-GlcNAcylation sites in APP(649-695) The second part of the aim is to analyze the transcript levels of APP, OGT, O-GlcNAcase (OGN) genes in Tg+/- brains, as well as those of other proteins involved in the APP metabolism (left side of the scheme). Preliminary data suggests that the mRNA levels of the APP, OGT and OGN genes are similar in the brains of tg+/- mice. Results: His-MBP-APP was successfully expressed and purified from E.Coli. The C-terminal APP (649-695) was produced through incubation of the fusion protein with recombinant TEV protease. His-MBP-APP represents a poor substrate for O-GlcNAc transferase, most likely because folding of His-MBP-APP obscures the target residues in the APP tail. Further O-GlcNAc labeling studies require free APP(649-695), and this work is ongoing. Characterization of O-glycosylation in collagen Aims: 1) The role of GLT25d1, a recently identified galactosyl transferase involved in glycosylation of collagen, was characterized by comparing the site specific glycosylation of collagen secreted from wild type osteoblasts and those in which the gene encoding for GLT25d1 was suppressed. The site-specific glycosylation changes derived from suppression of Glt25d1 were determined. The glycosylation status of immature (bifunctional) and mature (trifunctional, pyridinoline) cross-links in type I bone collagen and type II cartilage collagen was determined. In bone, the immature cross-links are modified with comparable amounts of G-(galactosyl) and GG-(glucosylgalactosyl-) glycoforms, while the dominant glycoform of the pyridinoline cross-links was determined to be G-Pyr. In cartilage type II collagen, both immature and mature cross-links were found to be mainly non-glycosylated. The glycosylation of the pyridinoline cross-links was found to change in an age-specific fashion, i.e. the levels of GG-Pyr relative to the levels of non-modified Pyr increase from fetal to young to old cartilage. In cartilage type II collagen, a novel type of cross-link modification was identified and partially characterized by LC-MS/MS. The immature cross-links typically emerge by formation of a NaBH4-reducible keto-immine bond between a hydroxylysine aldehyde from the N-telo or C-telo region and the &#949;-amino group of a specific hydroxylysine residue from the helical region. The reduction of the keto-immine bond is detectible by the characteristic +2 Da increase in MS. In contrast, this modification renders the immature cross-links non-reducible and results in a mass increase of +189 Da compared to the mass of the non-reduced cross-linker. This modification was found very abundant in fetal cartilage and to a lesser extent in young and mature cartilage. It is believed that it might be involved in collagen maturation. The molecular composition was partially derived from MS/MS data, based on specific fragmentation pattern of this modified cross-linker in MS/MS. Rev1-DNA Crosslinking. A methodology for the specific enrichment and MS analysis of protein-DNA crosslinks is currently being developed. A novel biotinylated disulfide-containing oligonucleotide was designed for crosslinking and subsequent specific enrichment prior to MS analyses. An offline HPLC purification approach to specifically isolate Rev1-DNA crosslinked peptides was developed. To increase specificity, offline HPLC of radioactive fractions (indicating a crosslink between the DNA and protein) was evaluated, pooled, digested, and analyzed by mass spectrometry. TSG6. For this work, we are investigating the identification of the residue in TSG-6 that crosslinks to hyaluronan. Oxidative Stress Radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In this work, we have investigated the sites of radicals trapped by DMPO on deoxyribonucleosides and proteins using LC/MS/MS. a. For proteins: DMPO adducts have been determined for LPO, MPO, and acetylcholinesterase. Isotopically DMPO was synthesized to help with the MS/MS fragmentation interpretation. Glyceraldehyde-3-phosphate dehydrogenase was identified as a possible protein target. b. For DNA: A DMPO adduct was identified on several bases from purified DNA as well as in cellular DNA. Radical formation of DNA is being extended to several systems for radical formation (Fiona and Suchandra).